Some cAMP stimulation of TNFRI release in exosome-like vesicles depended on two BIG2 AKAP sequences as well as the A-kinase RII B subunit. The presence of at least one cAMP phosphodiesterase (PDE) in the AKAP complex was likely. PDE activities (total, PDE3, and PDE4) were assayed in samples of HeLa cytosol and proteins precipitated from it by BIG1 or BIG2 antibodies. Each immunoprecipitate (IP) contained ca 4% of cytosolic PDE4 and 15-19% of PDE3 activity, essentially all of which was identified as PDE3A. PDE3A IPs contained both BIG1 and BIG2 which also appeared partially co-localized on confocal immunofluorescence microscopy. Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates human ADP-ribosylation factor (ARF) 1 and 3 by accelerating the replacement of ARF-bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Liquid chromatography MS/MS analysis of proteins that co-precipitated from HepG2 cells with BIG1 antibodies identified kinesin family member 21A (KIF21A), a plus-end-directed motor protein that moves cargo on microtubules away from the microtubule-organizing center. Reciprocal (IP) of endogenous proteins and microscopically apparent overlap of immunoreactive BIG1 with overexpressed GFP-KIF21A in the perinuclear region were consistent with KIF21ABIG1 interaction. Overexpression of full-length KIF21A and BIG1 and their fragments in HEK293 cells followed by reciprocal IP revealed that the C-terminal tail of KIF21A, with seven WD-40 repeats, may interact with structure in the C-terminal region of BIG1. Interfering with cyclic activation and inactivation of ARF1 by overexpressing constitutively active ARF1(Q71L) or dominant inactive ARF1(T31N) altered the distribution of BIG1 as well as its interaction with KIF21A. A requirement for ARF1 was confirmed by its selective depletion with siRNA. Unlike disruption of microtubules with nocodazole, selective inhibition of transport by depletion of KIF21A with specific siRNA altered BIG1 distribution without changing that of intrinsic Golgi membrane proteins. These newly recognized interactions of BIG1 and KIF21A should enable us to understand better the mechanisms through which, acting together, they may integrate local events in membrane trafficking with longer-range transport processes and to relate those processes to the diverse signaling and scaffold functions of BIG1. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.